We propose an alternate minimization algorithm for estimating the point-spread function (PSF) of a confocal laser scanning microscope and the specimen fluorescence distribution. A three-dimensional separable Gaussian model is used to restrict the PSF solution space and a constraint on the specimen is used so as to favor the stabilization and convergence of the algorithm. The results obtained from the simulation show that the PSF can be estimated to a high degree of accuracy, and those on real data show better deconvolution as compared to a full theoretical PSF model.